Apparatus for analyzing particles in urine and method thereof

ABSTRACT

An apparatus, intended for use in analyzing particles in urine is disclosed, that comprising: a sample distribution section for distributing urine samples to a first aliquot and a second aliquot; a first specimen preparing section for preparing a first specimen for measuring urinary particles, containing at least erythrocytes, by mixing a first stain reagent and the first aliquot; a second specimen preparing section for preparing a second specimen for measuring bacteria by mixing a second stain reagent and the second aliquot; and an optical detecting section comprising a light source for irradiating a light to a specimen being supplied, a scattered light receiving element for detecting scattered light emitted from the specimen, and a fluorescence receiving element for detecting fluorescence emitted from the specimen. A method intended for use in analyzing particles in urine is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from Japanese Patent Application2006-137511 filed on May 17, 2006, the disclosure of which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an apparatus for analyzing particles inurine and method thereof, and more specifically to an apparatus foranalyzing particles in urine for optically measuring and analyzingconstituents contained in urine with the use of flow cytometry.

BACKGROUND

General measurement items of particles in urine include erythrocytes,leukocytes, epithelial cells, casts and bacteria. Of these, the cast isproduced in such a manner that Tomm-Horsfall mucoproteins clotted andprecipitated in the renal tubule lumen under the presence of a smallamount of plasma protein (albumin) act as a substrate, and blood cellsand renal tubule epithelial cells are embedded therein. Casts as largeas several tens of μm or more are present and owe their name to the factthat they are formed using a cylinder or the renal tubule lumen as atemplate. (Presence of casts suggests that there was a temporaryocclusion with the renal tubule lumen, is considered important asfindings indicating substantial renal disorder, and especially thoseenclosing blood cells or epithelial casts have significant clinicalimportance.)

Further, epithelial cells consist of squamous cells and transitionalepithelium cells. Squamous cells have a circular or polygonal shape, areextremely thin and are created by detachment of a part of urinary tract.Meanwhile, transitional epithelium cells have diversified shapes such asa pear shape or a spinning top shape, and serve as component cells up torenal pelvis, urinary bladder and internal urethral opening. Their sizeranges from those as small as several tens μm to those as large as 100μm or more as represented by superficial cells.

Measurement of erythrocytes is important for judgment of presence orabsence of hemorrhage in the route from kidney glomerulus to the urinarytract, and is frequently noticed with urine samples from patients withrenal and urinary tract disorders, hemorrhagic disorders, leukemia orthe like. Although erythrocytes are normally approximately 8 μm in sizeand are disk-like cells having a concave shape on both sides, in mostcases, they present in the urine in a damaged form. In particular,erythrocytes derived from glomeruli are being deformed and are reducedin size. Erythrocytes with greater damage are hemolyzed and theircontents are eluted.

Leukocytes are frequently found in urine samples from patients withrenal infection, urinary tract infection, renal tuberculosis or thelike. Therefore, it is possible to detect inflammation and infection atearlier stage through measurements of leukocytes in the urine sample.Leukocytes are from about 6 to 14 μm in size. Measurement of bacteria isan examination to check presence or absence of infection. The bacteriainclude cocci and bacilli. Cocci are spherical bacteria from about 0.5to 2 μm in size, while bacilli are bacteria having a major axis in therange of about 2 to 10 μm. Cocci, if proliferated, result in aconglomeration of a chained shape representing an in-line moniliform orof a grape shape representing an irregularly and botryoidally-aggregatedones.

Conventionally, analysis of particles in urine has been performed byvisual inspection using a microscope in a general laboratory. With thismethod, a urine sample is first subjected to centrifugal separation andenriched, sediments thus obtained are in some cases stained and thenloaded on a microscope slide, and are subjected to classification andcounting under the microscope. By the microscope inspection, first,presence or absence of urinary particles is checked and status of theurine sample is grasped under low-power field (LPF) (×100), andclassification of each of constituents is performed under high-powerfield (HPF) (×400). Of measurement items, casts are small in number evenif appeared. However, detection of this item is clinically highly usefuland hence they are searched under low-power field (LPF). Other particlesare classified under high-power field (HPF), erythrocytes and leukocytesare searched under high-power field (HPF) and their count is reported.As mentioned, urinary particle examination has three factors—qualitativeexamination (for example, “++” for bacteria), quantitative examination(for example, “5 cells/HPF” for erythrocytes) and morphologicalexamination (for example, “presence of a poikilocyte is found” forerythrocytes).

For automation of urinary particle examination, an automatic microscopehas been proposed. As a flow-type automatic microscope, UA-2000(manufactured by Sysmex Corporation) is currently used. With thisdevice, a urine sample is introduced to a flat type flow cell withoutconcentration and images are taken and stored while it is flowingthrough the flow cell. The stored images are being sorted according tothe size of particles, and a user observes the images and classifiesthem to each particle.

For such automatic microscope method, one designed to classify particlesautomatically has been proposed recently. However, urinary particles arediversified in their morphology and many particles are being damaged,and therefore, classification of images taken with good accuracyaccompanies difficulties. It is particularly difficult to classifysmall-sized particles, such as erythrocytes (especially disruptederythrocytes), bacteria and crystals with good accuracy, and userintervention is needed for re-classification.

As an automatic classification apparatus for urinary particleexamination, a urinary particle measuring apparatus UF-100 based on theflow cytometer (manufactured by Sysmex Corporation) has been proposed.In this apparatus, urinary particles are stained by a stain reagent, anda scattered light signal and a fluorescence signal are combined toexecute classification of erythrocytes, leukocytes, epithelial cells,casts and bacteria. As for a classification reagent, a dye for stainingthe membrane and nucleus of each particle is used, and morphology ofurinary particles is maintained (see, for example, Japanese PatentLaid-Open No. 8-170960). As mentioned above, urinary particles arediversified in their morphology and many particles are being damaged,and it is difficult to execute classification with good accuracy only bya combination of scattered light signal intensity and fluorescencesignal intensity of a flow cytometer. Hence, a configuration is proposedwhich utilizes a combination of intensity of the scattered light signaland its pulse width, intensity of the fluorescence signal and its pulsewidth to allow for classification of each of urinary particles (see, forexample, U.S. Pat. No. 5,325,168). This urinary particle measuringapparatus based on the flow cytometer involves various ingenuities toallow for classification of urinary particles and presentation ofmorphology information. For example, information about origin of urinaryerythrocytes (derived from glomeruli or from glomeruli) is presentedthrough analysis of scattered light signals of erythrocytes (see, forexample, U.S. Pat. No. 6,118,522). This apparatus enables automaticclassification of urinary particles, thereby contributing greatly toautomation of urine examinations.

Even if an apparatus designed to analyze scattered light signal andfluorescence signal by various methods is used, there are samples whichhinder high-accuracy measurements. As a possible cause for this, it ismentioned that there are samples with which classification of bacteriais difficult. Bacteria, although considerably small in size, havediversified morphologies. For example, samples in which small-sizedbacteria, namely spherical bacteria not in a conglomeration form, aloneare dominant are mentioned. The apparatus mentioned above is composed sothat large-sized particles (casts, epithelial cells), medium-sizedparticles (leucocytes, erythrocytes) and small-sized particles(bacteria) are measured at one time, which means measurements oflarge-sized particles of several tens μm are necessary, and therefore,detection of bacteria of 1 μm or smaller in their size with goodaccuracy was difficult. Bacteria measurement in the urinary particleexamination is an examination to see whether or not bacteria appeared inan amount which could be confirmed by microscope inspection, and ingeneral, presence or absence of bacteria about 10⁴ cells/ml or more ischecked. If a bacteria concentration of 10⁴ cells/ml or more isavailable, cocci are proliferated and are mostly in a conglomerationform, which normally meets requirements of the urinary particleinspection. However, so far, samples with small-sized spherical bacterianot in a conglomeration form were occasionally overlooked.

On the other hand, samples with large-sized bacteria, namely, specimensin which spherical bacteria in a grown conglomeration or large-sizedbacilli appeared, the range of bacteria appearance is widened and insome cases entered into the range of appearance of erythrocytes andleukocytes. Further, even if spreading into erythrocyte and leukocyteappearance range does not occur, their appearance range overlaps withthat of mucus fibril, yeast-like fungi and sperms, thereby inviting, insome cases, erroneous results in the bacteria measurement.

In the meantime, although depending on the clinical purpose intended, ahigh-sensitivity examination is also required for the bacteria test ofurine. However, detection of fewer numbers of bacteria by visualexamination under a microscope is difficult particularly for small-sizedbacteria, and this is not used for examinations which needhigh-sensitivity. In this case, a cultivation test in which a specimenis cultured to be subjected to the examination is carried out in thebacteria laboratory separately from urinary particle inspection. Sincecultivation test needs considerable number of days for cultivation, itis proposed that high-sensitivity bacteria test be performed withoutcultivation.

Bacteria analysis utilizing flow cytometry, namely, a method in whichbacteria are stained by a stain reagent and are measured by a scatteredlight signal and a fluorescence signal, has been proposed. For example,European Patent Application Publication No. EP1136563 and U.S. PatentApplication Publication No. U.S. 2002/0076743 disclose a method formeasuring specimens such as urine containing foreign substances having asimilar size as that of bacteria with good accuracy with the use of adying reagent containing a cationic surfactant to allow for dissolutionof foreign substances other than bacteria.

SUMMARY

The scope of the present invention is defined solely by the appendedclaims, and is not affected to any degree by the statements within thissummary. A first apparatus, intended for use in analyzing particles inurine, embodying features of the present invention comprises: a specimenpreparing section for preparing one or more specimens by using a urinesample, a first stain reagent and a second stain reagent; an opticaldetecting section comprising a light source for irradiating the one ormore specimens with light, a scattered light receiving element fordetecting light scattered from the one or more specimens, and afluorescence receiving element for detecting fluorescence emitted fromthe one or more specimens; a first measurement section for measuringparticles in urine containing at least erythrocytes, based on scatteredlight and fluorescence detected by the optical detecting section; and asecond measurement section for measuring bacteria in urine, based onscattered light and fluorescence detected by the optical detectingsection.

A second apparatus, intended for use in analyzing particles in urine,for measuring bacteria contained in urine and urinary particlescontaining at least erythrocytes embodying features of the presentinvention comprises: a sample distribution section for distributingurine samples to a first aliquot and a second aliquot; a first specimenpreparing section for preparing a first specimen for measuring urinaryparticles, containing at least erythrocytes, by mixing a first stainreagent and the first aliquot; a second specimen preparing section forpreparing a second specimen for measuring bacteria by mixing a secondstain reagent and the second aliquot; an optical detecting sectioncomprising a light source for irradiating a light to a specimen beingsupplied, a scattered light receiving element for detecting scatteredlight emitted from the specimen, and a fluorescence receiving elementfor detecting fluorescence emitted from the specimen; and a temperatureregulating section for regulating a temperature of the first specimenpreparing section to a first temperature and regulating a temperature ofthe second specimen preparing section to a second temperature higherthan the first temperature.

A first method, intended for use in analyzing particles in urine,embodying features of the present invention comprises: a) preparing oneor more specimens by using a urine sample, a first stain reagent and asecond stain reagent; b) irradiating light to the one or more specimens,and detecting scattered light and fluorescence emitted from the one ormore specimens; c) measuring particles in urine containing at leasterythrocytes, based on scattered light and fluorescence detected fromone of the one or more specimens; and d) measuring bacteria in urine,based on scattered light and fluorescence detected from one of the oneor more specimens.

A second method, intended for use in analyzing particles in urine, formeasuring bacteria contained in urine and urinary particles containingat least erythrocytes, embodying features of the present inventioncomprises: a) distributing a urine sample to a first aliquot and asecond aliquot; b) preparing a first specimen for measurement of urinaryparticles containing at least erythrocytes, by mixing a first stainreagent and the first aliquot; c) preparing a second specimen formeasurement of bacteria by mixing a second stain reagent and the secondaliquot; d) detecting scattered light and fluorescence emitted from thefirst specimen by irradiating light to the first specimen; and e)detecting scattered light and fluorescence emitted from the secondspecimen by irradiating light to the second specimen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view explaining one embodiment of an apparatusfor analyzing particles in urine according to the present invention;

FIG. 2 is a drawing showing an outline of a functional composition of aspecimen preparing section and an optical detecting section of theapparatus for analyzing particles in urine;

FIG. 3 is a drawing showing a composition of the optical detectingsection;

FIG. 4 is a drawing showing a relationship between absorption wavelengthand absorbance of one example of a first stain reagent;

FIG. 5 is a drawing showing a relationship between absorption wavelengthand absorbance of one example of a second stain reagent;

FIG. 6 is a block diagram showing a whole composition of the apparatusfor analyzing particles in urine shown in FIG. 1;

FIG. 7 is a diagrammatic perspective view of a quantifying mechanism andthe specimen preparing sections of the apparatus for analyzing particlesin urine;

FIG. 8 is a drawing explaining the quantifying mechanism and thespecimen preparing section of the apparatus for analyzing particles inurine;

FIG. 9 is a flowchart (first half) showing urine analysis proceduresusing the apparatus for analyzing particles in urine relating to oneembodiment according to the present invention;

FIG. 10 is a flowchart (second half) showing urine analysis proceduresusing the apparatus for analyzing particles in urine relating to oneembodiment according to the present invention;

FIGS. 11( a) to 11(e) are drawings showing one example of a scattergramobtained by the apparatus for analyzing particles in urine relating toone embodiment according to the present invention;

FIG. 12 is a drawing showing one example of a scattergram of a bacteriasystem obtained by the apparatus for analyzing particles in urinerelating to one embodiment according to the present invention; and

FIG. 13 is a drawing showing one example of a scattergram of a bacteriasystem obtained by the apparatus for analyzing particles in urinerelating to one embodiment according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to the attached drawings, embodiments of the apparatus foranalyzing particles in urine relating to one embodiment according to thepresent invention will be explained in detail. FIG. 1 is a perspectiveview explaining a main unit of the apparatus for analyzing particles inurine relating to one embodiment according to the present invention anda personal computer attached thereto. In FIG. 1, a chassis foraccommodating components of the apparatus for analyzing particles inurine is omitted in part to facilitate good understanding.

Composition of Apparatus

In FIG. 1, an apparatus for analyzing particles in urine (main unit) Uincludes a specimen preparing section 2 for preparing a specimen, a racktable 4 for transferring a sample rack (test tube stand) 3, an opticaldetecting section 5 for detecting information about urinary particlesand bacteria from the specimen, and a circuit section 14. A supportstand 16 is provided to the chassis side face via an arm 15, and apersonal computer 13 is mounted thereon. The personal computer 13 isconnected to the circuit section 14 of the apparatus for analyzingparticles in urine U via LAN connection.

FIG. 2 is a drawing showing an outline of a functional composition ofthe specimen preparing section 2 and the optical detecting section 5. InFIG. 2, urine (sample) contained in a test tube T is sucked by a syringepump (not shown) using a suction pipe 17 and is dispensed to thespecimen preparing section by a sample distribution section 1. Thespecimen preparing section in the present embodiment is composed of aspecimen preparing section (first specimen preparing section) 2 u and aspecimen preparing section (second specimen preparing section) 2 b, thesample distribution section 1 distributes aliquots of quantified urine(sample) to each of the specimen preparing section 2 u and the specimenpreparing section 2 b.

To the urine aliquots in the specimen preparing section 2 u are mixedwith a dilute solution 19 u and a dyeing solution (stain reagent) 18 u,and dyeing is performed by dyes contained in the dyeing solution (stainreagent) 18 u. This stained specimen is used as a suspending solutionfor analyzing relatively large urinary particles (urinary sediments)such as erythrocytes, leukocytes, epithelial cells and casts. Meanwhile,to the urine aliquots in the specimen preparing section 2 b are mixed adilute solution 19 b and a dyeing solution (stain reagent) 18 b, anddyeing is performed by dyes contained in the dyeing solution (stainreagent) 18 b. This stained specimen is used as a suspending solutionfor analyzing bacteria.

With regard to two types of suspending solutions (specimens) prepared asmentioned above, the suspending solution (first specimen) of thespecimen preparing section 2 u is first introduced to the opticaldetecting section 5, forms a fine stream being wrapped by a sheathsolution in a sheath flow cell 51, and laser light is irradiatedthereto. Following this, in a similar fashion, the suspending solution(second specimen) of the specimen preparing section 2 b is introduced tothe optical detecting section 5, forms a fine stream in the sheath flowcell 51, and laser light is irradiated thereto. These operations arecarried out automatically by actuating driving units and solenoid valves(not shown) by controlling a microcomputer 11 (control apparatus), whichwill be described later.

FIG. 3 is a drawing showing a composition of the optical detectingsection 5. In FIG. 3, a condenser lens 52 focuses laser light irradiatedfrom a semiconductor laser 53, which acts as the light source, onto thesheath flow cell 51, and a collecting lens 54 focuses forward-scatteredlight of urinary particles onto a photodiode 55, which acts as ascattered light receiving element. Further, another collecting lens 56focuses side scattered light and side fluorescence of the particles ontoa dichroic mirror 57. The dichroic mirror 57 reflects side scatteredlight on a photomultiplier 58, which acts as a scattered light receivingelement and causes side fluorescence to be transmitted towards aphotomultiplier 59, which acts as a fluorescence receiving element.These optical signals are considered to reflect features of urinaryparticles. The photodiode 55, photomultiplier 58 and photomultiplier 59convert optical signals to electrical signals, and each outputs aforward scattered light signal (FSC), a side scattered light signal(SSC), and a side fluorescence signal (SFL), respectively. After beingamplified by a preamplifier (not shown), these outputs are subjected tothe next processing.

As for the light source, although a gas laser may be used in lieu of thesemiconductor laser, it is preferable to employ a semiconductor laserfrom the viewpoints of low costs, small-size and low power consumption,and employment of a semiconductor laser could reduce product costs andat the same time, allow realization of small-sized apparatus andelectrical power saving. Further, among semiconductors lasers, a redsemiconductor laser is preferably used due to low costs, long servicelife and stable supply by the manufacturers.

FIG. 6 is a block diagram showing a whole composition of the apparatusfor analyzing particles in urine U. In FIG. 6, the apparatus foranalyzing particles in urine U includes the above-mentioned sampledistribution section 1, specimen preparing section 2, and opticaldetecting section 5, an analog signal processing circuit 6 for executingamplification and filter processing of the output of the opticaldetecting section 5 for those being amplified by the preamplifier, anA/D converter 7 for converting the output of the analog signalprocessing circuit 6 to a digital signal, a digital signal processingcircuit 8 for executing a predetermined waveform processing for digitalsignals, memory 9 connected to the digital signal processing circuit 8,the microcomputer 11 connected to the analog signal processing circuit 6and the digital signal processing circuit 8, and a LAN adapter 12connected to the microcomputer 11. The personal computer 13 (analysissection) provided outside is LAN connected to the apparatus foranalyzing particles in urine U via this LAN adapter 12, and analysis ofdata acquired by the apparatus for analyzing particles in urine U iscarried out by the personal computer 13. The analog signal processingcircuit 6, A/D converter 7, digital signal processing circuit 8, andmemory 9 compose a signal processing circuit 10 for electric signalsbeing output by the optical detecting section 5.

Urinary Particle Measuring Reagent

As for reagents for measuring urinary particles, Japanese PatentLaid-Open No. 8-170960 provides detailed description. In one embodimentof the reagents, a dye for membrane staining is selected in order tostain even particles without nuclei. As for a reagent, an osmoticpressure compensation agent is added for the sake of prevention oferythrocyte hemolysis and acquirement of stable fluorescence intensity,and it is adjusted to 100 to 600 mOsm/kg in order to obtain osmoticpressure suited for classification and measurement. Cell membranes andnuclei (nuclear membrane) of urinary particles are stained by thisreagent. As for a stain reagent containing a dye for membrane staining,a condensed benzene derivative is used and, for example, NK-529 (tradename, manufactured by Hayashibara Biochemical Labs.) that is a cyaninedye may be used. Meanwhile, this stain reagent is designed to stainnuclear membrane as well as cell membranes. When such a stain reagent isused, for nucleated cells such as leukocytes and epithelial cells,dyeing intensity in the cellular cytoplasm (cell membrane) and the samein nucleus (nuclear membrane) are joined together, and this allows fordiscrimination of nucleated cells such as leukocytes and epithelialcells from urinary particles without nucleus such as erythrocytes or thelike.

Bacteria Analysis Reagent

As for reagents for measuring bacteria with good accuracy also forspecimens such as urine containing similar size foreign substances,European Patent Application Publication No. EP1136563 provides detaileddescription. In one embodiment of the reagents, a dye for nucleic acidstaining is used. As for stain reagents containing a dye for nucleusstaining, for example, a cyanine dye represented by the followingchemical structure (1) and disclosed by U.S. Patent ApplicationPublication No. U.S. 2002/0076743 may be used.

Of these, a cyanine system dye represented by the following chemicalstructure (2) may be used preferably.

In this case, it is preferable that a stain reagent (first stainreagent) to be added to a urine sample for measurement of urinaryparticles containing at least erythrocytes contains a dye for membranestaining, while a stain reagent (second stain reagent) to be added tothe urine sample for measurement of bacteria contains a dye for nucleicacid staining. Since urinary particles contain those having no nucleussuch as erythrocytes, it is possible to detect urinary particlesincluding those having no nucleus when the first stain reagent containsa dye for membrane staining. Further, when the second stain reagentcontains a dye for-nucleus staining, nuclei of bacteria are stainedeffectively, and it is possible to make a measurement of the bacteriawith good accuracy even if they are small in size.

Further, it is preferable that, for the first stain reagent and thesecond stain reagent, a peak of absorption wavelength is present inproximity to luminescence wavelength of the semiconductor laser. Byselecting the peak of absorption wavelength of the first stain reagentand the second stain reagent so that it may present in proximity to theluminescence wavelength of the semiconductor laser, it becomes possibleto make a measurement of stained urinary particles and bacteria by thesemiconductor laser. FIG. 4 is a drawing showing a relationship betweenabsorption wavelength and absorbance of one example of the first stainreagent of this sort (stain reagent containing NK-529 (trade name,manufactured by Hayashibara Biochemical Labs.)), in which a peak ofabsorption wavelength exists at 640 nm in the vicinity of theluminescence wavelength (635 nm) of a red semiconductor laser. FIG. 5 isa drawing showing a relationship between absorption wavelength andabsorbance of one example of the second stain reagent (stain reagentcontaining a cyanine dye represented by the chemical structure (2) anddisclosed in U.S. Patent Application Publication No. U.S. 2002/0076743),in which a peak of absorption wavelength exists at 636 nm in thevicinity of the luminescence wavelength (635 nm) of a red semiconductorlaser. Although FIG. 5 shows changes in absorbance for cases wherereagent temperatures are 15° C. and 35° C., it is noticed from this thatthere is no big change in absorbance of reagents insofar as thetemperature is around room temperature.

For bacteria measurement reagents, a cationic surfactant is included inorder that dyes pass through a membrane thereby promoting quick stainingand for the sake of shrinkage of foreign substances such as mucus fibriland debris of erythrocytes. In this case, since it is possible to givedamage to cell membranes of bacteria by the surfactant, nucleic acid ofbacteria can be stained effectively by the dye contained in the secondstain reagent. As a result, measurement of the bacteria can be performedwith further improved accuracy after a dyeing processing in a shortperiod of time. Meanwhile, in a case where a surfactant is mixed to thesecond aliquot, it is preferably configured so that measurement ofbacteria takes place upon completion of measurement of urinaryparticles. Since the surfactant is contained in the second specimen, ifmeasurement of urinary particles is made after bacteria measurement, thesurfactant is mixed into the first specimen due to carry-over of thespecimen, membrane of the urinary particles containing erythrocytes isdamaged, and there is a possibility that measurements of the urinaryparticles are eventually affected. However, if it is configured so thatmeasurement of bacteria is made after completion of measurement ofurinary particles, mixing of the surfactant into the first specimen canbe prevented and measurement of urinary particles can be done with goodaccuracy.

According to the present embodiment, the first specimen for measurementof urinary particles containing at least erythrocytes and the secondspecimen for measurement of bacteria are prepared from one urine sample,respectively, and urinary particles containing at least erythrocytes aremeasured by the first specimen and bacteria are measured by the secondspecimen. With this configuration, one analysis apparatus is able tomake a measurement of urinary particles containing at least erythrocytesand bacteria with high accuracy, respectively. In addition, since theoptical detecting section is commonly used by the first specimen and thesecond specimen, composition of the apparatus can be simplified, therebyreducing products costs and downsizing the apparatus.

FIG. 7 is a diagrammatic perspective view of the quantifying mechanismand the specimen preparing sections of the apparatus for analyzingparticles in urine relating to the present embodiment, and FIG. 8 is adrawing explaining them. According to the present embodiment, a samplingvalve 30, which is used regularly, is employed as the quantifyingmechanism for distributing a predetermined amount of urine sample to thespecimen preparing section (first specimen preparing section) 2 u andthe specimen preparing section (second specimen preparing section) 2 b.This sampling valve 30 includes two disk-like fixed elements and amovable element being sandwiched by the fixed elements, and the movableelement is turned by a motor 31.

The sampling valve 30 is equipped with two discs 30 a, 30 b made ofalumina ceramics superimposed with each other. A flow path is formedinside of the disks 30 a, 30 b for circulation of the sample, the flowpath is isolated when one disk 30 b is turned around the center axisthereof, thereby quantifying the sample. Such sampling valve 30 iscomposed so as to form one unit with the specimen preparing section 2 bvia a hydraulic cassette 33 having a flow path 32 for the specimeninside. In other words, the sampling valve 30, the hydraulic cassette33, and the specimen preparing section 2 b are disposed so as tothermally form one unit being in close contact with each other, and areconfigured so that the temperature of the sampling valve 30 may becomealmost identical with that of the specimen preparing section 2 b. In themeantime, the specimen preparing section 2 u is fixed by a bolt 35 to amounting plate 34 fixed to the chassis while a predetermined clearance Sis provided, and therefore, the specimen preparing section 2 u is beingalmost thermally isolated from the sampling valve 30 and the specimenpreparing section 2 b.

The specimen preparing section 2 u and the specimen preparing section 2b are heated by heaters 36 u, 36 b, respectively, each composing atemperature regulating section. With this configuration, the temperatureof the specimen preparing section 2 u for preparing the first specimenis regulated to a first temperature and at the same time, thetemperature of the specimen preparing section 2 b for preparing thesecond specimen is regulated to a second temperature higher than thefirst temperature. In particular, the specimen preparing section 2 u isregulated to attain approximately 35±2° C. and the specimen preparingsection 2 b is regulated to attain approximately 42±2° C., that ishigher than the former. The higher the temperature of a specimen beingset, the faster the prescribed portion (membrane or nucleus) oferythrocytes or bacteria contained in the specimen is stained, therebyshortening the time for measurements. On the other hand, erythrocytesare easily damaged at high-temperatures, and if the temperature is settoo high, correct measurement is not possible. Hence, it is possible tomake measurements of urinary particles containing erythrocytes togetherwith bacteria with good accuracy if the temperature of the secondspecimen for measuring bacteria with higher heat resistance compared toother urinary particles is regulated to be higher than the temperatureof the first specimen for measuring urinary particles; in other words,the specimen preparing section 2 u and the specimen preparing section 2b are regulated to a temperature suitable for the measurement,respectively. Meanwhile, the temperature of the specimen preparingsection 2 u and the specimen preparing section 2 b may be measured by,for example, a thermistor. It is then possible to regulate the specimenpreparing section 2 u and the specimen preparing section 2 b to theprescribed range of the temperature, respectively, by ON-OFF control ofthe heaters 36 u, 36 b based on the results of measurements thusobtained.

Further, if the sampling valve 30 and the specimen preparing section 2 bare composed to thermally form one unit, it is possible to preventcooling of a specimen, which has been temperature-regulated by thesampling valve 30, when being supplied to the specimen preparing section2 b. This can reduce losses related to the temperature regulation. Inthis case, for a specimen being supplied to the specimen preparingsection 2 u which is kept at a lower temperature than the specimenpreparing section 2 b, its temperature could be reduced naturally whilebeing supplied from the sampling valve 30, if so configured that theflow path of the specimen passes through the clearance S.

Analysis Procedures

Next, referring to flow charts shown in FIG. 9 to FIG. 10, urineanalysis procedures using the apparatus for analyzing particles in urinerelated to one embodiment according to the present invention will beexplained.

First, specimen information such as sample number, patient informationsuch as name, age, gender, clinical specialty associated with the samplenumber, and measurement items being controlled by a host computer areobtained from the host computer (Step S1). A measurement executioninstruction is then given by input means such as a keyboard or a mouseof the personal computer 13 (Step S2). Upon receiving this instruction,the sample rack 3 in which are set test tubes T each containing thesample is transferred by the rack table 4 to a predetermined suctionposition (Step S3). The test tube T is turned at this suction positionand a barcode printed on an ID label, which is pasted on the outercircumference of the test tube T, is being read (Step S4). The samplenumber of the sample is then known, which is then verified with thesample information acquired in step S1, and measurement items of thesample can be identified.

Then, the suction pipe 17 goes down, a front edge of the suction pipe 17is inserted into the sample in the test tube T, and the sample islightly sucked and discharged repeatedly in this state so that thesample may be stirred (Step S5). After being stirred, a predeterminedamount (800 μL) of the sample is sucked, 150 μL and 62.5 μL of thesample is each dispensed by the sampling valve 30 to the specimenpreparing section 2 u for preparing a specimen for the measurement ofurinary particles containing at least erythrocytes (SED) and to thespecimen preparing section 2 b for preparing a specimen for themeasurement of bacteria (BAC) contained in the urine, respectively (StepS7 and Step S11).

To the specimen preparing section 2 u are quantified and dispensed apredetermined amount of a dyeing solution (stain reagent) and a dilutesolution together with the sample (Step S8 and Step S9). Similarly, tothe specimen preparing section 2 b are quantified and dispensed apredetermined amount of a dyeing solution (stain reagent) and a dilutesolution together with the sample (Step S12 and Step S13). The specimenpreparing section 2 u and the specimen preparing section 2 b are beingheated by the heaters 36 u, 36 b to a predetermined temperature,respectively, and stirring of the specimen is carried out by a propellertype stirrer (not shown) (Step S10 and Step S14). Meanwhile, the dilutesolution dispensed to the specimen preparing section 2 u in Step S9includes a surfactant, which gives damage to cell membranes, therebyallowing efficient dyeing of nuclei of bacteria.

Following this, a sheath solution is sent to the sheath flow cell 51 ofthe optical detecting section 5 (Step S15), the specimen for themeasurement of urinary particles (SED) is then introduced to the opticaldetecting section 5, and a fine stream (sheath flow) wrapped by thesheath solution is formed in the sheath flow cell 51 (Step S16). Laserbeam from the semiconductor laser 53 is irradiated to the sheath flowthus formed (Step S17). The reason why the measurement of urinaryparticles is performed first is that since a bacteria measurementspecimen includes a surfactant, if urinary particle measurement isperformed after bacteria measurement, the surfactant is mixed into thespecimen for urinary particle measurement due to carry-over of thespecimen, membrane of the urinary particles containing erythrocytes isdamaged, thereby affecting the measurement of the urinary particles insome cases.

Forward-scattered light, fluorescence, and side-scattered light ofurinary particles generated by the laser beam irradiation are receivedby the photodiode 55, photomultiplier 59, and photomultiplier 58,respectively, converted to electric signals, and are output as aforward-scattered light signal (FSC), a fluorescence signal (FL), and aside-scattered light signal (SSC) (Steps S18 to 20). These outputs areamplified by the preamplifier (Steps S21 to 23).

Upon completion of measurement by the specimen for measuring urinaryparticles (SED), bacteria in the urine are measured subsequently usingthe specimen prepared in Step S14. In this case, in a similar fashion asobserved in Steps S15 to 23, a forward-scattered light signal (FSC) anda fluorescence signal (FL) are output by the optical detecting section 5used in the measurement of urinary particles, and amplified.

The forward-scattered light signal (FSC), fluorescence signal (FL), andside-scattered light signal (SSC) thus amplified are converted todigital signals in the signal processing circuit 10 (see FIG. 6) and atthe same time, subjected to the predetermined waveform processing (StepsS24 to 27), and are transmitted to the personal computer 13 via the LANadapter 12. In the meantime, “FLH” in Step S25 is high-sensitivity oneobtained by amplifying the fluorescence signal (FL) with higher gain,and “FLL” in Step S26 is low-sensitivity one obtained similarly byamplifying the fluorescence signal (FL) with lower gain.

Then, raw data of the urinary particles (SED) are generated in thepersonal computer 13 (Step S28) and at the same time, a scattergram isgenerated based on the data (Step S29). Then, clustering of thescattergram prepared by algorithm analysis is performed (Step S30), andthe number of particles is counted for every cluster (Step S31).

For bacteria, in a similar fashion, the forward-scattered light signal(FSC) and the fluorescence signal (FL) being amplified are converted todigital signals in the signal processing circuit 10 and at the sametime, subjected to the predetermined waveform processing (Steps S32 to34). In the meantime, “FSCH” in Step S32 is high-sensitivity oneobtained by amplifying the forward-scattered light signal (FSC) withhigher gain, and “FSCL” in Step S33 is low-sensitivity one obtainedsimilarly by amplifying the forward-scattered light signal (FSC) withlower gain.

Then, they are transmitted to the personal computer 13 via the LANadapter 12. Raw data of the bacteria (BAC) are generated in the personalcomputer 13 (Step S35), and a scattergram is generated based on the data(Step S36). Then, clustering of the scattergram prepared as mentioned byalgorithm analysis is performed (Step S37), and the number of particlesis counted for every cluster (Step S38). Results of the measurementobtained as mentioned above are displayed on a display which is adisplay means of the personal computer 13 (Step S39).

As measurement results of the urinary particles (SED), scattergrams aregenerated from each of signals of forward-scattered light,side-scattered light, and fluorescence. FIG. 11( a) is a scattergram inwhich the horizontal axis represents fluorescence intensity(low-sensitivity) (FLL) and the vertical axis representsforward-scattered light intensity (FSC). Epithelial cells (EC) andleukocytes (WBC), which are large cells having nuclei, appear in aregion of strong fluorescence signal intensity. Majority of epithelialcells are larger in cell size than leukocytes and appear in a regionwhere fluorescence intensity is stronger than that of leukocytes, whilethe range of appearance of some small-sized epithelial cells overlapswith that of leukocytes. In order to distinguish the both, aside-scattered light signal is used. FIG. 11( b) is a scattergram inwhich the horizontal axis represents side-scattered light intensity(SSC) and the vertical axis represents forward-scattered light intensity(FSC). Since epithelial cells appear in a region where side-scatteredlight intensity is stronger than leukocytes, epithelial cells areidentified from this scattergram.

FIG. 11( c) is a scattergram in which the horizontal axis representsfluorescence intensity (high-sensitivity) (FLH) and the vertical axisrepresents forward-scattered light intensity (FSC) and shows a regionwhere fluorescence intensity is low. Erythrocytes (RBC) have no nucleiand therefore are found in regions where fluorescence intensity is low.Some crystals (X'TAL) appear in regions of erythrocytes appearance, andtherefore, a side-scattered light signal is used for confirmation ofappearance of crystals. FIG. 11( b) is a scattergram in which thehorizontal axis represents side-scattered light intensity (SSC) and thevertical axis represents forward-scattered light intensity (FSC). Withcrystals, the center of distribution of side-scattered light intensityis not constant, crystals appear in regions where the intensity is high,and therefore, discrimination from erythrocytes is performed from thisscattergram.

FIG. 11( d) is a scattergram in which the horizontal axis representsfluorescence width (FLLW) and the vertical axis represents fluorescencewidth 2 (FLLW2). FLLW indicates a width of a fluorescence signal tocapture particles in which cell membranes are stained and FLLW2indicates a width of a stronger fluorescence signal such as nuclei. Asshown in the drawing, FLLW of casts (CAST) is greater and FLLW2 of castswith contents (P. CAST) is greater. Further, casts without contents(CAST) appear in regions where FLLW2 is low. Here, a width of a signalreflects length of time during which an optical signal is being detectedon a pulse-like signal waveform where the vertical axis representssignal intensity and the horizontal axis represents time.

With another result of measurements of bacteria, scattergrams aregenerated from forward-scattered light signal and fluorescence signal.FIG. 11( e) is a scattergram in which the horizontal axis representsfluorescence intensity (B-FLH) and the vertical axis representsforward-scattered light intensity (high-sensitivity) (B-FSC). In urinaryparticle measurements, as shown by the scattergram in FIG. 11( c), arange of bacteria appearance overlaps with that of mucus fibril (MUCUS),yeast-like fungi (YLC), and sperms (SPERM). However, with bacteriameasurement, foreign substances such as mucus fibril and debris oferythrocytes are caused to constrict by a bacteria measurement reagent,and therefore, there is such a region where only bacteria appearindependently. In addition, since measurements are made withapproximately 10 times improved sensitivity compared to urinary particlemeasurements, small-sized bacteria can also be detected with highaccuracy, thereby ensuring accurate results of bacteria measurements.

In the present embodiment, since bacteria are solely measured, reductionin reliability of the automatic classification apparatus resulting frompresence of bacteria can be suppressed (bacteria are diversified intheir size ranging from large ones to small ones, and there is noregularity in their distribution, they cross over on the image withother constituents such as leukocytes and erythrocytes, thereby makingit difficult to facilitate correct segmentation). Even if a casejudgment is made as to whether or not an instruction for re-examination(review) by a microscope, the reason for low reliability of measurementresults can be attached at issuance of a re-examination instruction.

FIG. 11( e) shows a standard appearance region of bacteria (BACT), whilethe appearance region is depending on types of bacteria. FIG. 13 is anexample of measurements of a sample in which a large quantity of cocciappeared and chained. In this scattergram, the region where bacteria(BACT) appeared is distributed with an angle of approximately 45° withregard to the horizontal axis (fluorescence intensity). In other words,bacteria (BACT) appeared in regions where forward-scattered lightintensity (FSC) is high. With such samples, in the urinary particlemeasurement (SED), bacteria would appear in wider ranges, and eventuallyappear even in ranges, of erythrocyte appearance regions, whereforward-scattered light intensity (FSC) is low. With these samples,reliability of erythrocyte measurement is low. Meanwhile, FIG. 12 showsan example of measurement of samples containing bacilli. In thisscattergram, the region where bacteria (BACT) appeared is distributedwith a lower angle (approximately 5 to 10 degrees) with regard to thehorizontal axis (fluorescence intensity). Namely, bacteria (BACT)appeared in a region where forward-scattered light intensity (FSC) islow. With such specimens, even if bacilli is contained in a largeamount, forward-scattered light intensity (FSC) in the bacteriaappearance region is lower than that of the erythrocyte appearanceregion, and erythrocyte measurement is not affected by bacteria.Similarly, influences of bacteria on leukocyte (WBC) appearance regionin the urinary particle (SED) measurement can be confirmed from bacteriadistribution in the bacteria measurement (BAC). Judgment of presence orabsence of influences on measurement results of other particlesaccording to the tendency of bacteria distribution as mentioned above iscarried out by algorithm analysis by the personal computer 13 (analysissection), and results of judgment are displayed on the display togetherwith other measurement results in the Step S39.

As stated above, measurement reliability of other particles is confirmedby assuming bacteria appearance regions in the urinary particle (SED)measurement based on bacteria distribution obtained from bacteriameasurement (BAC). Accordingly, it is possible to make judgment whetheror not an instruction for re-examination (review) by a microscope shouldbe given according to the distribution of bacteria measurement (BAC),thereby reducing false positive judgments and giving appropriateinstructions for re-examinations. Furthermore, judgments ofre-examination resulting from impossibility of fractionation withhigh-reliability could be reduced.

Further, it is possible to configure to allow for measurements ofbacteria in urine when bacteria concentration in the urine is 5×10³cells/ml or more. Specifically, measurement of bacteria withconcentrations as low as 5×10³ cells/ml is made possible by setting themeasurement time longer or by increasing the amount of measurementspecimen. With the configuration in which measurement of bacteria inurine is possible when bacteria concentration in the urine is 5×10³cells/ml or more, bacteria measurement with sensitivity required forurine examination can be performed.

As shown above, in the apparatus for analyzing particles in urinerelating to one embodiment according to the present invention, such aconfiguration is employed that a specimen is prepared for a urine sampleusing a first stain reagent and a second stain reagent, urinaryparticles containing at least erythrocytes are measured by a firstmeasurement means, and bacteria are measured by a second measurementmeans. Therefore, it is now possible to make measurements with highaccuracy by one analysis apparatus urinary particles containing at leasterythrocytes and bacteria, respectively.

The optical detecting section of the apparatus for analyzing particlesin urine relating to the present embodiment is configured so thatreceiving of forward-scattered light and side-scattered light emitted bythe specimen is possible and the first measurement means is capable ofmeasuring urinary particles based on both scattered lights. Therefore,it becomes possible, by using side-scattered light, to distinguish, forexample, epithelial cells and leukocytes, or epithelial cells andbacteria, thereby improving accuracy of automatic classification (forexample, false positive judgments are reduced) and at the same time,reducing the number of specimens to be subjected to re-examination undera microscope.

The foregoing detailed description and accompanying drawings have beenprovided by way of explanation and illustration, and are not intended tolimit the scope of the appended claims. Many variations in the presentlypreferred embodiments illustrated herein will be obvious to one ofordinary skill in the art, and remain within the scope of the appendedclaims and their equivalents.

What is claimed is:
 1. A method, intended for use in analyzing particlesin urine, comprising: distributing a urine sample into a first aliquotand a second aliquot; preparing a first specimen by mixing the firstaliquot and a first stain reagent; preparing a second specimen by mixingthe second aliquot and a second stain reagent which is different fromthe first stain reagent; irradiating light to the first specimen, anddetecting first scattered light and first fluorescence emitted from thefirst specimen; irradiating light to the second specimen, and detectingsecond scattered light and second fluorescence emitted from the secondspecimen; measuring particles in urine, the particles containing atleast erythrocytes, based on the first scattered light and the firstfluorescence detected from the one or more specimen; and measuringbacteria in urine, based on the second scattered light and the secondfluorescence detected from the second specimen.
 2. The method of claim1, further comprising a step for judging effects of bacteria in resultsof the measuring of particles in urine, based on results of themeasuring of bacteria in urine.
 3. The method of claim 1, wherein:forward-scattered light and side-scattered light emitted from thespecimen are detected from the first specimen; and the measuring ofparticles in urine is based on the detected forward-scattered light andthe detected side-scattered light.
 4. The method of claim 1, wherein thefirst stain reagent contains a dye for staining membrane and the secondstain reagent contains a dye for staining nucleus.
 5. The method ofclaim 1, wherein in preparing the second specimen, a surfactant is mixedwith the second aliquot.
 6. The method of claim 5, wherein the detectingof the second scattered light and the second fluorescence emitted fromthe second specimen is executed after the detecting of the firstscattered light and the first fluorescence emitted from the firstspecimen.
 7. The method of claim 2, wherein in preparing the secondspecimen, a temperature of the second specimen is regulated to atemperature higher than the temperature of the first specimen.
 8. Amethod, intended for use in analyzing particles in urine, for measuringbacteria contained in urine and urinary particles containing at leasterythrocytes comprising: a) distributing a urine sample to a firstaliquot and a second aliquot; b) preparing a first specimen formeasurement of urinary particles, the particles containing at leasterythrocytes, by mixing the first aliquot and a first stain reagent; c)preparing a second specimen for measurement of bacteria by mixing thesecond aliquot and a second stain reagent which is different from thefirst stain reagent; d) detecting first scattered light and firstfluorescence emitted from the first specimen by irradiating light to thefirst specimen; and e) detecting second scattered light and secondfluorescence emitted from the second specimen by irradiating light tothe second specimen.
 9. A method, intended for use in analyzingparticles in urine, comprising: distributing a urine sample into a firstaliquot and a second aliquot; preparing a first specimen by mixing thefirst aliquot and a first stain reagent; preparing a second specimen bymixing the second aliquot and a second stain reagent which is differentfrom the first stain reagent; irradiating light to the first specimen,and detecting first scattered light and first fluorescence emitted fromthe first specimen; irradiating light to the second specimen, anddetecting second scattered light and second fluorescence emitted fromthe second specimen; measuring particles in urine, the particlescontaining at least erythrocytes, based on the first scattered light andthe first fluorescence detected from the first specimen; measuringbacteria in urine, based on the second scattered light and the secondfluorescence detected from the second specimen; displaying a firstscattergram showing a distribution of at least erythrocytes based on aresult of the measuring particles in urine; and displaying a secondscattergram showing a distribution of at least bacteria based on aresult of the measuring bacteria in urine.